Nucleic acid testing (NAT) assays provide powerful tools for the rapid detection and/or quantification of target nucleic acids. As such, NAT assays are commonly used to detect the presence of organisms in a sample, e.g., in patient samples in a clinical setting, in food samples, in environmental samples and the like. NAT assays are also commonly used in diagnostic settings, e.g., to detect genetic polymorphisms, genetic repeats, insertions, deletions, or the like, or altered gene expression, as indicative of a condition such as a disease or disorder.
In many situations, it is desirable to obtain quantitative information regarding the amount of a target nucleic acid sequence in a given sample. For example, quantitative nucleic acid assays, e.g., amplification assays, can be used to detect the presence and/or amount of a pathogen-specific target sequence present in a biological sample to determine whether the sample is infected with the pathogen, and/or to monitor the progression or severity of the infection. Quantitative nucleic acid assays can also be useful for monitoring the state of a cell or tissue by monitoring the amount of a marker nucleic acid sequence present in the cell or tissue, or for quantifying the amount of a specific DNA element, for example a repeat or a transposable element, present in a sample.
Quantitative nucleic acid amplification reactions can be used for quantifying the relative and/or absolute amount of target nucleic acid sequences present in a sample. Such methods have become highly advanced and sensitive, such that only a few copies of target nucleic acid can be detected in a sample. Due to the highly sensitive nature of quantitative nucleic acid amplification reactions, in order to avoid false positives, false negatives, overestimation of target or product quantity, or underestimation of target or product quantity, extreme care must be taken when choosing appropriate internal controls. In addition to considerations regarding the specificity of internal controls (e.g., internal control templates, primers and/or probes), considerations regarding intrinsic features of control nucleic acid sequences, including, e.g., hairpins, A/T runs with very low annealing temperatures, G/C runs with very high annealing temperatures that do not make the nucleic acids amenable to amplification and/or probe hybridization exist. Additionally, it may be desirable to use the same internal control template sequence, primer set, and probe in a variety of multiplex reactions, for a variety of target sequences. However, many nucleic acid sequences are amenable to amplification and/or probe hybridization under a narrow set of reaction conditions, and are not sufficiently robust to be used as internal controls under a wide variety of reaction conditions.
Thus, one skilled in the art will appreciate the complicated nature of identifying a robust combination of an internal control sequence, primers, and probes for performing an internal control to monitor nucleic acid amplification. Moreover, one skilled in the art will appreciate that extensive empirical validation is often performed to verify that a polynucleotide template sequence, primer pair, probe, or combination thereof will function as a viable/adequate internal control for quantitative nucleic acid amplification.